Yoneshima H, Miyawaki A, Michikawa T, Furuichi T, Mikoshiba K
Department of Molecular Neurobiology, University of Tokyo, Japan.
Biochem J. 1997 Mar 1;322 ( Pt 2)(Pt 2):591-6. doi: 10.1042/bj3220591.
To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P2R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 microM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 microM. These Ca2+-dependent alterations of Ins(1,4,5)P3 binding to both Ins(1,4,5)P3R types were reversible. Scatchard analyses revealed that Ca2+ changed the affinity of both Ins(1,4,5)P3R types but not the total number of Ins(1,4,5)P3-binding sites. The Kd values of Ins(1,4,5)P3R1 for Ins(1,4,5)P3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 microM free Ca2+. In contrast, Ins(1,4,5)P3R3 exhibited an affinity for Ins(1,4,5)P3 with Kd values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 microM free Ca2+. These results indicate that (1) both Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ regulates the binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 in opposite manners.
为阐明1型和3型肌醇-1,4,5-三磷酸受体（分别为Ins(1,4,5)P3R1和Ins(1,4,5)P3R3）之间的功能差异，我们研究了Ca2+对这两种类型Ins(1,4,5)P3R配体结合特性的影响。我们从cDNA在昆虫卵巢Sf9细胞中表达全长人Ins(1,4,5)P3R1和Ins(1,4,5)P3R3，其膜部分用于Ins(1,4,5)P3结合测定。Ins(1,4,5)P3与Ins(1,4,5)P3R1和Ins(1,4,5)P3R3的结合受到Ca2+的不同调节。随着游离Ca2+（[Ca2+]）浓度增加，Ins(1,4,5)P3与Ins(1,4,5)P2R1的结合减少，而与Ins(1,4,5)P3R3的结合增加。在[Ca2+]范围从小于1 nM到大于10 μM时观察到Ins(1,4,5)P3与Ins(1,4,5)P3R1结合的改变。Ins(1,4,5)P3R1的Ins(1,4,5)P3结合的EC50为100 nM Ca2+。相反，Ins(1,4,5)P3与Ins(1,4,5)P3R3的结合在高[Ca2+]时改变，EC50值为872 nM，且在100 nM至10 μM之间急剧变化。Ins(1,4,5)P3与这两种类型Ins(1,4,5)P3R的结合的这些Ca2+依赖性改变是可逆的。Scatchard分析表明，Ca2+改变了这两种类型Ins(1,4,5)P3R的亲和力，但未改变Ins(1,4,5)P3结合位点的总数。Ins(1,4,5)P3R1对Ins(1,4,5)P3的Kd值在游离Ca2+为3 nM时为78.5 nM，在游离Ca2+为1.4 μM时为312 nM。相反，Ins(1,4,5)P3R3对Ins(1,4,5)P3的亲和力在游离Ca2+为3 nM时Kd值为116 nM，在游离Ca2+为1.4 μM时为62.2 nM。这些结果表明：（1）Ins(1,4,5)P3R1和Ins(1,4,5)P3R3都至少有两种亲和力状态；（2）Ca2+调节这些状态之间的相互转化；（3）Ca2+以相反方式调节Ins(1,4,5)P3与Ins(1,4,5)P3R1和Ins(1,4,5)P3R3的结合。
